This invention relates to the formation of stable branch migration structures and the various applications of these structures.
The displacement of one strand of a double-stranded nucleic acid by another single strand with an identical nucleotide sequence is a well documented aspect of DNA or RNA replication and genetic recombination in vivo. Branch points are found in nucleic acids that are undergoing this kind of strand displacement where two strands compete for base pairing interactions with complementary sequences of a third strand. The movement of branch points along the strands of the nucleic acid, branch migration, does not require the action of specific enzymes or proteins.
The phenomenon of branch migration in vitro in the renatured molecules of terminally repetitious, circularly permuted bacteriophage DNA was first reported by Lee, Davis, and Davidson [JMB 48: 1-22 (1970)]. Branched nucleic acid structures suitable for the study of strand displacements can be constructed in vitro using various hybridization conditions.
Branch migration has been exploited to form or resolve DNA-RNA hybrids. In solutions without formamide, a DNA strand will displace RNA from a DNA-RNA hybrid. This reaction is the basis of a homogeneous nucleic acid hybridization assay developed by Vary et al. of Allied Corporation [Nuc. Acids Res. (1987) 15, 6883-6897 and U.S. Pat. No. 4,795,701]. This assay involves RNase digestion of the displaced RNA strand, conversion of AMP to ATP and detection of the product of the conversion by chemiluminescence using luciferase. Vary""s method is not applicable to DNA cloning.
In concentrated formamide solutions, a DNA strand may be displaced by RNA to form an R-loop [see Thomas, M., White, R. L, and Davis, R. W. (1976) Proc. Nat. Acad. Sci., USA 73, 2294-2298]. Conceptually, R-loop formation is analogous to the displacement of one DNA strand from the end of a duplex. Regions of double-stranded DNA can take up complementary RNA sequences to form R-loops under conditions where RNA:DNA hybrids are more stable [Casey, J. and Davidson, N. (1977) NAR 4: 1539-1552]. The enrichment of specific DNA sequences has been accomplished using buoyant density sedimentation to select for R-loop structures containing these sequences. The technique of R-loop formation has not been patented. To date, applications of R-looping procedures involve partial denaturation of the target DNA and have not yielded products that can be directly cloned into standard cloning vectors.
D-loop formation that is analogous to R-loop formation can occur between a DNA strand and a superhelical DNA duplex [Radding, C. M., Beattie, K. L., Holloman, W. K., and Wiegand, R. C. (1977) J. Mol. Biol. 116, 825-839]. This reaction depends upon the superhelical free energy and will, thus, not take place with linear DNA molecules. No cloning technology based on this observation has been described. D loop formation in superhelical DNA has been used for specific cleavage [Corey, D. R., Pei, D. and Schultz, P. G. (1989) J. Am. Chem. Soc. 111, 8523-8525].
A method has been developed which uses RecA-coated strands to overcome the limitation of D-loop formation to superhelical DNA molecules [Rigas, B., Welcher, A. A., Ward, D. C., and Weissman, S. M. (1986) Proc. Natl. Acad. Sci., USA 83, 9591-9595]. Labeling of the single-stranded probe with biotinylated nucleotides facilitates purification of the D-loop products of this reaction by affinity chromatography. DNA hybrids that contain biotinylated nucleotides have lower melting temperatures than unmodified DNA hybrids, i.e., biotin has a destabilizing effect on the helix [Langer, et al. (1981) Proc. Natl. Acad. Sci. USA 78: 6633-6637]. In this method D-loop formation requires implementation of a pretreatment step to facilitate D-loop formation. Moreover, this procedure does not result in products that are directly clonable into existing DNA cloning vectors.
A short DNA strand hybridized to a longer DNA strand will also be rapidly displaced by a homologous, but longer, overlapping strand in vitro [see Green, C. and Tibbetts, C. (1981) Nuc. Acids Res. 9, 1905-1918]. This observation forms the basis of diagnostic assays for DNA or RNA sequences that are based on branch migration and DNA strand displacement, described in Collins et al. [Mol. and Cell. Probes 2: 15-30 (1988)], Vary et al. [Clin. Chem. 32: 1696-1701 (1986)], U.S. Pat. Nos. 4,766,064 [Williams et al. (1988), Allied Corp.], 4,766,062 [Diamond et al (1988), Allied Corp.], and 4,795,701 [Vary et al (1988), Allied Corp.] and European Patents 0167238 A1 [Collins et al (1985), Allied Corp.] and 0164876 A1 (Collins et al. (1985) Allied Corp.].
In these assays, a partially double-stranded probe complex is prepared with a detectable label on one of the two strands. This probe complex is then incubated with a sample containing target nucleic acids (i.e., double-stranded nucleic acids that are at least partially homologous to the single-stranded portion of the probe complex) under appropriate hybridization conditions. The target nucleic acids hybridize to the single-stranded portion of the probe complex and undergo branch migration to release the labeled probe strand. The amount of labeled strand released is proportional to the amount of target DNA in a sample. Thus, this assay involves the use of a pre-formed partially double-stranded probe complex to promote branch migration and relies upon the transient nature of the branch migrated structure and the total release of the labeled probe strand.
In these assays, the efficiency of the strand displacement reaction could be enhanced by the addition of volume excluding reagents, such as polyethers, or by pretreatment of the target with Rec A proteins. Others have also noted that the Rec A protein promotes branch migration that proceeds unidirectionally in the 3xe2x80x2- greater than 5xe2x80x2 direction [Cox, M. M. and Lehman, I. R. (1981) Proc. Natl. Acad. Sci. USA 78: 6018-6022]. The diagnostic assays developed by the Allied Corp. use preformed duplexes to promote displacement and do not relate to the development of genetic engineering techniques or to the stabilization of the branch migration structure.
Thus, the phenomenon of branch migration initiated by the formation of a stable hybrid has been described in the literature. Although the formation of branched or looped structures has been used for identification, purification, and enrichment of DNA sequences, this technique has not been applied to the development of a directly clonable product. Moreover, stabilization of branch migration structures would enhance the efficiency of procedures that involve collection or identification of these entities. Simple methods for preparing stable branch migration structures have not been reported by others.
Experiments described more than 20 years ago showed that the substitution of bromine at position C5 of pyrimidines leads to increased duplex stability [Michelson et al.(1967) Prog. Nuc. Acid Res. and Mol. Bio. 6: 84-141]. Radding et al. [J. Biol. Chem. 116: 2869-2876 (1962)] showed that dG-BrdC is a more thermally stable base pair than dG-dC. In another study, poly dl:poly BrdC had a melting temperature 26xc2x0 C. higher than poly dI:poly dC [Inman and Baldwin (1964) J. Mol. Bio. 8: 452-469], and it was further shown that poly BrdC displaced poly dC from a poly dI:poly dC duplex to form a new duplex with poly dI [Inman J. Mol. Bio. 9: 624-637 (1964)]. These observations have not been applied to the stabilization of branch migration structures by displacer strands containing modified nucleotide bases.
Tatsumi and Strauss [Nuc. Acids Res. 5: 331-347 (1978)] labeled DNA with bromodeoxyuridine (BrdUrd) in vivo in human lymphoid cells and observed a high degree of branch migration after isolation and shearing of the DNA. These workers suggested that this high level of branch migration reflected the increased stability of helices containing BrdUrd and the trapping of the branch migration configuration. Their results further suggest that once formed, halogen-substituted branch migration structures are relatively stable. Tatsumi and Strauss did not investigate the phenomenon of branch migration in vitro, did not use synthetic oligo- or polynucleotides labeled with halogenated nucleotides, i.e., pre-modified displacer strands, in their experiments, and could not use the branch migration structures resulting from their experiments for purposes of cloning or mutagenesis.
Currently, specific DNA fragments derived from genomic DNA are usually identified using Southern blot analysis of the restriction enzyme digestion products of that genomic DNA. Southern blot analysis of DNA is a multi-step procedure that generally involves the use of radioisotopes and autoradiography for fragment identification following electrophoresis, transfer of the electrophoresced products to a nitrocellulose or nylon membrane, and hybridization of the transferred products to sequence-specific DNA probes. Procedures that provide for simultaneous labeling and identification of specific DNA fragments would be significantly simpler, faster, and cheaper than Southern blot analyses and would have a significant impact on the field of genetic engineering.
Labeling of DNA sequences has come to imply the incorporation of modified nucleotides at internal or terminal positions. Often this is accomplished enzymatically using nucleotides labeled with small compounds such as biotin, sulfhydryl groups, mercury, allylamine, and digoxigenin. Enrichment or purification of these labeled nucleic acids can be accomplished by affinity chromatography. For example, biotinylated DNA, including D loops, can be selectively bound and eluted from solid matrices bearing avidin or streptavidin groups, as has been reported by Ward and his co-workers [Langer, op. cit.]. Similarly, DNA labeled with sulfhydryls can be purified by affinity chromatography on mercurated agarose and mercurated DNA can be purified based on its affinity for sulfhydryl groups. The enrichment of mRNA from total RNA populations can be accomplished based on the affinity of the polyA tails on the messengers for oligo-dT matrices. Following their purification, these enriched or purified nucleic acid sequences are often further subjected to a series of procedures that render them clonable. Labeling procedures that permit sequence enrichment or purification by affinity chromatography and then the direct cloning of these enriched or purified fractions would have significant advantages over existing techniques.
The identification or enrichment of a specific subset of nucleotide sequences or of DNA fragments in isolated genomic DNA or total populations of RNA for the express purpose of cloning those sequences or fragments has been accomplished by a variety of methods that include, but are not limited to those involving the selection of DNA fragments capable of undergoing branch migration, R-loop, or D-loop formation. Selective cloning of fragments in a population has been achieved based on restriction endonuclease cleavage sites, especially those that recognize a unique site [Brown, N. L. and Smith, M. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3213-3216], and size [Thomas, M., Cameron, J. R. and Davis, R. W. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 4579-4583]. Although these strategies have been used widely and successfully to clone a wide variety of genes, they are not universal and have limited specificity.
Another application of genetic engineering techniques relates to the modification of genetic material so as to add or delete nucleotide bases, such as for therapeutic purposes. Efforts to either replace, inactivate, or modify genetic material are currently in progress. In higher eukaryotic organisms, the approaches taken have, thus far, required that the agents used to bring about these changes be incorporated into various plasmid vectors that contain all or part of various viral genomes. Site-directed gene replacement in lower eukaryotes, such as yeasts, has been accomplished. A serious deterrent to the therapeutic manipulation of human genetic material in vivo relates to the lack of a suitable, benign vector system. The ability to perform targeted delivery and incorporation of genetic material into chromosomal DNA without using a viral vector would represent a major advance in the field of gene therapy.
Green and Tibbetts [op. cit.] expressed an interest in using branched DNA structures for in vitro site-directed mutagenesis and, in fact, were able to use stable D-loop structures in superhelical DNA for target deletion mutagenesis [Proc. Natl. Acad. Sci. USA 77: 2455-2459 (1980)]. They were unable to achieve this goal with linear target DNA molecules using the branch migrated structures they obtained from in vivo labeling of cells with BrdUrd (see Green and Tibbetts, 1981, op. cit.) due to the short half-lives of their branched structures.
Site-specific genetic manipulation has been described [Capecchi, M. R. (1989) Science 244, 1288-1292] where a small proportion of the DNA which becomes integrated into the host genome is directed, by homologous recombination, to the desired target DNA. Unfortunately, the additional integration events occur at random and may inactivate or activate genes leading to deleterious consequences. Sequences of DNA capable of initiating triple-helix formation have been reported. [Francois, J. C., Saison-Behmoaras, T., Thoung, N. T. and Helene, V. (1989) Biochemistry 28, 9617-9619; Povsic, T. J. and Dervan, P. B. (1989) J. Am. Chem. Soc. 111, 3059-3061]. The utilization of triple helix formation as an adjunct to site-specific genetic manipulation is unknown in the art.
In its most general terms our invention relates to techniques which increase the probability of certain types of reactions proceeding in a desired manner. Our techniques promote specificity of reaction and provide
simple methods for preparing stable branch migration structures.
procedures for simultaneous labeling and identification of specific DNA fragments that are significantly simpler, faster, and cheaper than Southern blot analyses.
labeling procedures that permit sequence enrichment or purification by affinity chromatography and selective cloning.
a method of performing targeted delivery and incorporation of genetic material into chromosomal DNA without using a viral vector.
Branch migration is the process by which a single strand of nucleic acid is inserted and replaces at least a portion of one strand of a nucleic acid duplex. Branch migration is a useful technique for the sequence-dependent attachment (capture) of an oligodeoxynucleotide duplex containing a single-stranded tail into the end of a deoxynucleotide molecule, or of the sequence dependent incorporation of an oligodeoxynucleotide into a deoxynucleotide molecule at a location other than at its end. Existing methods require the formation of a stable hybrid prior to the initiation of branch migration; our process allows initiation and formation of a branch migrated complex without prior stabilization. We have achieved this result by stabilizing the resulting branch migrated complex concurrently with formation or thereafter, or both.
Our specific attachment procedure may be used (A) to label a particular fragment for detection without blotting and subsequent hybridization, (B) to mark a particular fragment for affinity chromatography, (C) to facilitate cloning by introducing a new 5xe2x80x2 or 3xe2x80x2 overhang compatible with a restriction endonuclease site in a cloning vector, or (D) for other purposes which will become apparent from the present disclosure.
The various methods and materials of our invention require a displacer entity which is at least partially complementary to and is capable of binding to a target. Both the displacer and target are oligo- or polynucleotide sequences, either synthetic or naturally occurring. Our novel displacer may be used as a single stranded entity in certain embodiments of our invention; in other embodiments it is utilized as a partially double stranded entity hybridized to a linker strand.
One embodiment of our invention provides a novel displacer-linker duplex and an improved method of attaching a deoxynucleotide displacer sequence to a strand of a target deoxynucleotide duplex. The oligo- or polydeoxynucleotide displacer-linker duplexes of our invention consist of two strands, a displacer strand and a linker strand. The displacer strand contains a sequence of nucleotides at least partially complementary to the linker strand and a sequence at least partially complementary to one strand of a recipient polydeoxynucleotide duplex.
The sequence-dependent attachment (capture) of an oligodeoxynucleotide duplex containing a single-stranded tail can be influenced by branch migration into the end of a DNA molecule. Our novel oligo- or polydeoxynucleotide displacer-linker duplex is capable of initiating branch migration at the end of a recipient polydeoxynucleotide duplex without the prior formation of a stable hybrid with such recipient polydeoxynucleotide duplex. More particularly, these novel duplexes can hybridize to and initiate branch migration at a restriction endonuclease cleavage site, in particular adjacent to a 3xe2x80x2 or 5xe2x80x2 single stranded extension on a recipient polydeoxynucleotide duplex.
Substitution of one or more of the nucleotides in the portion of the displacer strand which is complementary to the recipient increases DNA-DNA hybrid stability. Oligonucleotides containing the modified nucleotide displace non-modified nucleotide containing strands from the ends of duplexes. In the case of 3xe2x80x2 or 5xe2x80x2 overhangs the rate of displacement is of the same order of magnitude as the nucleation reaction of DNA reassociation.
We also provide a displacer which is not hybridized to a linker strand and which is capable of initiating triple helix formation. This class of displacers comprises
1. a first sequence which is capable of initiating triple helix formation, and which comprises
a) at least six consecutive pyrimidine bases or
b) at least seven bases where at least six of the bases are pyrimidine bases and the seventh base is guanine, and
2. a second sequence proximate to such first sequence which is
a) complementary to and runs antiparallel to the second strand of the recipient duplex and
b) which is capable of initiating branch migration proximate to the triple helix.
We further disclose displacers which are nuclease resistant and the method of modifying a recipient duplex to confer nuclease resistance to the duplex. These displacers contain at least one moiety attached to a terminus of the oligo- or polynucleotide, which moiety confers endonuclease resistance to the terminus to which it is attached.
We have found it desirable to modify at least one of the nucleotides in the displacer strand which is at least partially complementary to one strand of the recipient polydeoxynucleotide duplex. The nucleotide is modified in a manner that increases the stability of the hybrid displacer-recipient duplex.
In addition to stability increasing modifications, we have found it useful to incorporate modified nucleotides in the displacer or linker which permit detection of the displacer-recipient hybrid or its isolation by affinity chromatography.
Another aspect of our invention is the hybrid structure formed when the displacer or displacer-linker duplex is attached to the target. Where the hybrid is the result of the attachment of the target to our novel displacer-linker duplex, the linker strand is preferably covalently linked to one of the strands of the recipient duplex.
In a preferred version of our invention, the hybrid structure containing the attached displacer sequence of single stranded deoxynucleotide is stabilized, most desirably by the presence of at least one modified nucleotide in the displacer strand.
We also disclose a labelled hybrid structure. This labelled hybrid structure incorporating our displacer-linker is useful in many biochemical procedures, such as, for example, to facilitate capture of the displacer-recipient hybrid by affinity chromatography, to label one end of an cloned deoxynucleotide insert in a vector, to facilitate restriction endonuclease mapping of an insert, to facilitate selectively cloning a recipient polynucleotide duplex and to facilitate isolation of clones of contiguous polydeoxyribonucleotides. The hybrid incorporating the displacer is useful in affinity chromatography and to facilitate 1] detection of specific oligo- or polynucleotides and 2] site specific genetic manipulation
Our methods involve the stabilization of a non stable complex between one strand of a recipient polydeoxynucleotide sequence and a displacer sequence of single stranded DNA where the displacer sequence is at least partially complementary to such strand of a recipient polydeoxynucleotide sequence. The complex may be stabilized by
a) the presence of at least one modified nucleotide in the displacer strand,
b) forming a DNA triplex between the displacer sequence and the recipient duplex,
c) providing a displacer strand comprising a nucleotide sequence and a sequence specific DNA binding moiety that does not significantly melt the recipient DNA duplex at the site to which it attaches,
d) attaching the displacer to a linker prior to or concurrent with attachment to the target duplex and thereafter covalently attaching the linker to the second strand of the target duplex, or
e) a combination of procedures a) and d) or c) and d).